Control prl sv40 renilla reporter vector

Cells were plated at a density of 50,000 cells per well in 24 well plates. Cells were co-transfected with a control pRL-SV40 Renilla Reporter Vector (Promega) to normalize luciferase readings. HEK293 cells and LNK cells were transfected using Lipofectamine 2000 (Invitrogen). LNK cells were transfected with 1000 ng of pGL3 construct and 50 ng of Renilla control using a ratio of 5 μl Lipofectamine to 1 μg DNA. 293 cells were transfected with 200 ng of pGL3 construct and 10 ng of Renilla control using a 0.6 μl Lipofectamine to 1 μg DNA for 293. EL-4 cells were transfected with 1500 ng pGL3 construct and 100 ng Renilla control using TrueFect-Max (United BioSystems, Herndon, VA, USA) at a ratio 2.5 μl TrueFect to 1 μg DNA. After 48 hours of incubation, cells were lysed and assayed for luciferase activity using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. Measurement of the firefly luciferase activity of the Ly49 promoter constructs was normalized relative to the activity of the Renilla luciferase produced by the pRL-SV40 control vector in order to control for differences in transfection efficiency, and each construct was tested in triplicate in at least three independent experiments